Endothelial nitric oxide synthase (eNOS) is an important mediator of penile erection. Appropriate subcellular trafficking and protein-protein interactions are necessary for proper eNOS function. Among the most important eNOS-protein interactions that may affect eNOS regulation in the vasculature including the penile vascular bed, are eNOS binding with caveolin-1 and heat shock protein (Hsp)90. While caveolin-1 facilitates eNOS trafficking to caveolae and proper organization of the eNOS signaling complex, it interferes with subsequent biological actions of eNOS. Hsp90 facilitates eNOS activation and keeps coupled eNOS activity. Currently very little is known about the role and regulation of eNOS-protein interactions in the regulation of the enzyme's activity and its functional relevance in the vasculature including the penile vascular bed. The overall goal of the proposal is to investigate the molecular mechanisms of eNOS regulation in the penis by eNOS-protein interactions, and its physiological significance in penile erection. The investigation will involve evaluation of eNOS interactions with caveolin-1 and HspQO in a mouse dyslipidemic model as a paradigm for this investigation. We will first evaluate whether eNOS interactions with caveolin-1 and HspQO are impaired in hypercholesterolemia-induced erectile dysfunction (ED) under flaccid or erect conditions, in parallel with a defect in eNOS function. Next, we will evaluate whether preserving eNOS-protein interactions preserves eNOS function and erectile function. This hypothesis will be tested in dyslipidemic mice treated with L-4F, an apolipoprotein-A-l mimetic, which positively influences eNOS function in part by maintaining an optimal eNOS-protein interaction. Third, we will evaluate whether increased superoxide production by NAD(P)H oxidase and uncoupled eNOS underlies impaired eNOS-protein interaction and ED in the hypercholestero- lemic mouse. This will be tested in hypercholesterolemic mice with and without apocynin-induced inhibition of NAD(P)H oxidase. Elucidation of the molecular mechanisms of eNOS regulation is crucial for a better understanding of NO-mediated responses in vascular tissues under physiologic and pathophysiologic circumstances. The proposed investigation is expected to elucidate a novel molecular mechanism of eNOS regulation in the penis and its physiological significance in erection physiology. At the same time, it may suggest a specific eNOS regulatory pathway as a possible new molecular target for treating ED. [unreadable] [unreadable] [unreadable]